Virulence factors contributing to invasive activities of Serratia grimesii and Serratia proteamaculans

© 2015, Springer-Verlag Berlin Heidelberg. Previously, we have shown that facultative pathogens Serratia grimesii and Serratia proteamaculans are capable to invade eukaryotic cells provided that they synthesize intracellular metalloprotease grimelysin or protealysin, respectively (Bozhokina et al. in Cell Biol Int 35(2):111–118, 2011). Noninvasive Escherichia coli transformed with grimelysin or protealysin gene became invasive, indicating that the protease is a virulence factor. Here we elucidated involvement of other virulence factors in the invasion of S. grimesii and S. proteamaculans. Under similar experimental conditions, the amount of S. proteamaculans internalized within human carcinoma HeLa cells was fivefold higher than that of S. grimesii. In accord with this, in S. proteamaculans, high activities of pore-forming hemolysin ShlA and extracellular metalloprotease serralysin were detected. In S. grimesii, activity of toxin ShlA was not detected, and the serralysin activity of the bacterial growth medium was very low. We also show that iron depletion strongly enhanced invasive activity of S. proteamaculans, increasing activities of hemolysin ShlA and serralysin, but did not affect S. grimesii properties. These results show that the invasive activity of S. proteamaculans is maintained, along with protealysin, by hemolysin and serralysin. On the other hand, grimelysin is so far the only known invasion factor of S.grimesii

Purification and characterization of the proteinase ECP 32 from Escherichia coli A2 strain

The proteinase previously described as an unidentified component of E. coli A2 extracts which hydrolyses actin at a new cleavage site (Khaitlina et al. (1991) FEBS Lett. 279, 49) was isolated and further characterized. A chromatographic method of proteinase purification was developed by which a purity of more than 80% was attained. The enzyme was identified as a single, 32 kDa polypeptide (ECP 32) by SDS-PAGE and non-denaturing electrophoresis as well as by ion-exchange chromatography and gel filtration. The N-terminal sequence of ECP 32 was determined to be: AKTSSAGVVIRDIFL. The activity of ECP 32 is inhibited by o-phenanthroline, EDTA, EGTA and zincone. The EDTA-inactivated enzyme can be reactivated by cobalt, nickel and zinc ions. Based on these properties ECP 32 was classified as a metalloproteinase (EC 3.4.24). Limited proteolysis of skeletal muscle actin between Gly-42 and Val-43 was observed at enzyme substrate mass ratios of 1:25 to 1:3000. Two more sites between Ala-29 and Val-30, and between Ser-33 and Ile-34 were cleaved by ECP 32 in heat- or EDTA-inactivated actin. Besides actin, only histones and DNA-binding protein HU were found to be substrates of the proteinase, confirming its high substrate specificity. Its molecular mass, N-terminal sequence and enzymatic properties distinguish ECP 32 from any known metalloproteinases of E. coli, and we therefore conclude that it is a new enzyme

The hemolytic propertiesof clinical isolates of Morganella morganii

Morganella morganii is a gram-negative bacterium from the Enterobacteriaceae family which causes a wide range of clinical infections sometimes with fatal consequences. It is known that more than 50% of isolates of M. morganii from clinical specimens have hemolytic activity that increase their virulence. Pore-forming toxins (PFT) represent the most common group of cytotoxic proteins which contribute the delivering of the bacterial proteins into host cells, loss of nutrients and ions by eukaryotic cells, as well as the exit of bacteria from phagosome into cytosol. In this study we investigated the hemolytic activity of two M. morganii strains. It has been shown that hemolytic activity for strain of M. morganii 4 is 3 times higher than for strain of M. morganii 1. The maximum hemolytic activity is observed in LB medium but synthesis of hemolysins is higher in synthetic urine. Finally, the PCR-analysis of 5 hypothetical hemolysin genes has shown that strain M. morganii 1 does not contain homologous of α-hemolysin from E. coli that may explain the observed differences in hemolytic activity of the investigated strains

Physico-chemical properties of actin cleaved with bacterial protease from E. coli A2 strain

The 36 kDa fragment of actin molecule obtained with the protease from E. coli A2 strain [(1988) FEBS Lett. 228, 172] was shown to begin with Val-43 and retain the COOH-terminal amino acid residues of the parent molecule. The E. coli protease split actin preserves the NH2-terminal part of the polypeptide chain as well as the native conformation of actin molecule. However, the E. coli protease split actin failed to polymerize in 0.1 M KCl, suggesting that integrity of actin molecule between Gly-42 and Val-43 is crucial for actin polymerization. © 1991

Hypertrophic Stimulation Increases β-actin Dynamics in Adult Feline Cardiomyocytes

The myocardium responds to hemodynamic stress through cellular growth and organ hypertrophy. The impact of cytoskeletal elements on this process, however, is not fully understood. While α-actin in cardiomyocytes governs muscle contraction in combination with the myosin motor, the exact role of β-actin has not been established. We hypothesized that in adult cardiomyocytes, as in non-myocytes, β-actin can facilitate cytoskeletal rearrangement within cytoskeletal structures such as Z-discs. Using a feline right ventricular pressure overload (RVPO) model, we measured the level and distribution of β-actin in normal and pressure overloaded myocardium. Resulting data demonstrated enriched levels of β-actin and enhanced translocation to the Triton-insoluble cytoskeletal and membrane skeletal complexes. In addition, RVPO in vivo and in vitro hypertrophic stimulation with endothelin (ET) or insulin in isolated adult cardiomyocytes enhanced the content of polymerized fraction (F-actin) of β-actin. To determine the localization and dynamics of β-actin, we adenovirally expressed GFP-tagged β-actin in isolated adult cardiomyocytes. The ectopically expressed β-actin-GFP localized to the Z-discs, costameres, and cell termini. Fluorescence recovery after photobleaching (FRAP) measurements of β-actin dynamics revealed that β-actin at the Z-discs is constantly being exchanged with β-actin from cytoplasmic pools and that this exchange is faster upon hypertrophic stimulation with ET or insulin. In addition, in electrically stimulated isolated adult cardiomyocytes, while β-actin overexpression improved cardiomyocyte contractility, immunoneutralization of β-actin resulted in a reduced contractility suggesting that β-actin could be important for the contractile function of adult cardiomyocytes. These studies demonstrate the presence and dynamics of β-actin in the adult cardiomyocyte and reinforce its usefulness in measuring cardiac cytoskeletal rearrangement during hypertrophic stimulation

Ca-dependent binding of actin to gelsolin

Khaitlina S, Hinssen H. Ca-dependent binding of actin to gelsolin. FEBS LETTERS. 2002;521(1-3):14-18.Ca2+ of 0.3-1.0 muM induces both the exposure of tryptic cleavage sites within the gelsolin molecule inaccessible in the Ca-free conformation, and binding of one actin monomer to the N-terminal half of gelsolin. On the other hand, gelsolin-induced enhancement of pyrene actin fluorescence was observed only above 50 muM Ca2+, and a ternary actin/gelsolin complex preformed in 200 muM Ca2+ was stable only above 30 muM Ca2+. These results provide direct evidence for a Ca-induced transitions from closed to open conformation of the gelsolin molecule in the range of 3 x 10(-7) to 10(-6) M Ca2+. They also suggest that Ca2+ > 10(-5) M is required to stabilize actin-actin contacts in the 2:1 actin/gelsolin complex. (C) 2002 Federation of European Biochemical Societies. Published by Elsevier Science B.V. All rights reserved

Conformational changes in actin induced by its interaction with gelsolin.

Actin cleaved by the protease from Escherichia coli A2 strain between Gly42 and Val43 (ECP-actin) is no longer polymerizable when it contains Ca2+ as a tightly bound cation, but polymerizes when Mg2+ is bound. We have investigated the interactions of gelsolin with this actin with regard to conformational changes in the actin molecule induced by the binding of gelsolin. ECP-(Ca)actin interacts with gelsolin in a manner similar to that in which it reacts with intact actin, and forms a stoichiometric 2:1 complex. Despite the nonpolymerizability of ECP-(Ca)actin, this complex can act as a nucleus for the polymerization of intact actin, thus indicating that upon interaction with gelsolin, ECP-(Ca)actin undergoes a conformational change that enables its interaction with another actin monomer. By gel filtration and fluorometry it was shown that the binding of at least one of the ECP-cleaved actins to gelsolin is considerably weaker than of intact actin, suggesting that conformational changes in subdomain 2 of actin monomer may directly or allosterically affect actin-gelsolin interactions. On the other hand, interaction with gelsolin changes the conformation of actin within the DNase I-binding loop, as indicated by inhibition of limited proteolysis of actin by ECP and subtilisin. Cross-linking experiments with gelsolin-nucleated actin filaments using N,N-phenylene-bismaleimide (which cross-links adjacent actin monomers between Cys374 and Lys191) reveal that gelsolin causes a significant increase in the yield of the 115-kDa cross-linking product, confirming the evidence that gelsolin stabilizes or changes the conformation of the C-terminal region of the actin molecule, and these changes are propagated from the capped end along the filament. These results allow us to conclude that nucleation of actin polymerization by gelsolin is promoted by conformational changes within subdomain 2 and at the C-terminus of the actin monomer

Virulence factors contributing to invasive activities of Serratia grimesii and Serratia proteamaculans

© 2015, Springer-Verlag Berlin Heidelberg. Previously, we have shown that facultative pathogens Serratia grimesii and Serratia proteamaculans are capable to invade eukaryotic cells provided that they synthesize intracellular metalloprotease grimelysin or protealysin, respectively (Bozhokina et al. in Cell Biol Int 35(2):111–118, 2011). Noninvasive Escherichia coli transformed with grimelysin or protealysin gene became invasive, indicating that the protease is a virulence factor. Here we elucidated involvement of other virulence factors in the invasion of S. grimesii and S. proteamaculans. Under similar experimental conditions, the amount of S. proteamaculans internalized within human carcinoma HeLa cells was fivefold higher than that of S. grimesii. In accord with this, in S. proteamaculans, high activities of pore-forming hemolysin ShlA and extracellular metalloprotease serralysin were detected. In S. grimesii, activity of toxin ShlA was not detected, and the serralysin activity of the bacterial growth medium was very low. We also show that iron depletion strongly enhanced invasive activity of S. proteamaculans, increasing activities of hemolysin ShlA and serralysin, but did not affect S. grimesii properties. These results show that the invasive activity of S. proteamaculans is maintained, along with protealysin, by hemolysin and serralysin. On the other hand, grimelysin is so far the only known invasion factor of S.grimesii